This invention concerns a method for stabilizing human or animal platelets without the use of aldehydes or other fixative agents for determining multiple platelet parameters in reference controls using electronic instrumentation, as well as diluents therefor. As a result of modern technological advances, instrumentation systems for enumeration of cellular biological components for clinical diagnostic purposes has increased, and this has created a need for improvement of quality control products.
To make the platelet reference controls, the platelets are removed from the blood by centrifugation, washed with buffered saline, and then "fixed" with agents such as glutaraldehyde, formaldehyde, pyruvic aldehyde, or the like. The aldehyde-reacted platelets are then suspended in a buffer.
The suspended platelets are not stable and will aggregate under a number of circumstances. In addition, the aldehyde-reacted platelets gradually change shape, shrinking in size as they age.
The aldehyde-reacted platelets are intended to be used in particle counters which are affected by particle size. Thus, the reference control should ideally contain platelets which are as similar as possible in size to the platelet particles in normal human blood. It is apparent that aggregation will affect the count and size of the particles in the reference controls. It is also apparent that any shrinking or swelling which occurs also will impair the value of the control, since the platelets are no longer the same size as platelets in fresh human blood.
Several human platelet reference controls are currently marketed. However, improvement is needed with respect to long term stability in the measurement of mean platelet volume and size distribution width.
One such instrumentation system is the Coulter.RTM. S-Plus hematology system which counts and examines the count and volume distribution of human platelets in whole blood specimens. Similar type instruments are manufactured by other companies for this purpose. Specifications as to electronic calibration are well defined to the operator of such instruments. Any human platelet reference control must satisfy all of the criteria that are measured on human patient specimens. The reference control needs to simulate as closely as possible that of normal fresh whole blood specimens. It must also conform to properly set thresholds as to the count mode, correctly balanced apertures, current, amplification gain settings, and responsiveness of the system as to all functional aspects.
Mean platelet volume distribution analysis has gained recognition recently as a useful measurement for clinical application. Human platelet size distribution width can now be calculated from specifically measured parameters. Each of the measured parameters lends itself to improvement in examining the log normal distribution of human platelets. Also, changes and shifts in the mode result in erroneous counts.
The common cause of count discrepancies is aggregation. Aggregation of platelets creates doublets that count in the white blood cell mode resulting in unreliable quality control measures. To the extent that platelets undergo aggregation, the aggregated platelet cells appear and are counted as white cells on the automated instrument. Since the number of platelets present are about 30 times the number of white cells present in fresh blood, even if only a small percentage of platelets aggregate, this can cause unreliable results with respect to the white cell count, especially after treatment with the usual fixing agents.
The adhesiveness of platelets also leads to false counts. The adhesion of platelets to "foreign" surfaces has been measured by a variety of methods. The principle of all of these tests is the same. The decrease in platelet count which occurs when blood is allowed to contact a "foreign" surface for a standarized period of time is determined. This decrease is indicative, in part, of the number of platelets which have adhered to the foreign surface, and this value, expressed as a percentage of the original platelet count, has been called adhesiveness. However, it is known that some of the loss of platelets is also due to platelet aggregation. Adhesiveness of platelets, although dependent upon either calcium or magnesium ion is not as selective in this regard as the adhesiveness of the polymorphonucleocytes.
Platelets participate in primary hemostasis by forming aggregates at the site of the injured blood vessels. The agent which is ultimately responsible for platelet aggregation is probably adenosine diphosphate (ADP), which may be derived from the injured tissues and erythrocytes or released from the platelets themselves by, among others, collagen, thrombin and epinephrine. In some patients with bleeding disorders, and in normal patients following an ingestion of some drugs, platelet aggregation by one or more of these agents may be impaired. This impairment of platelet aggregation may be the cause of the prolonged bleeding time which is often obtained in these patients.
Another discrepancy in platelet count is brough about by poor performance of stability until the expected expiration date of the control. Attempts to stabilize human platelets have proved to be extremely difficult. One major problem is that of disintegration of the platelet membrane. Fixatives have been used as a source of controlling disintegration. When platelet membranes disintegrate, they cause debris, thereby resulting in erroneous counts. New methods through improved computer technology for curve filling of raw data are beset by these problems.
Our invention discloses a method of processing human or animal platelets and stabilization without the use of fixatives such as glutaraldehyde which lends itself to eliminate the aforementioned discrepancies.
In U.S. Pat. No. 4,198,206 to Ryan (1980), which is a continuation-in-part of U.S. Pat. No. 4,160,644 (1979), a method is described for preparing a control from platelets which do not aggregate, and which have the same size as platelets in human blood, and maintain their size for at least 6 months. This control is a suspension of aldehyde-reacted platelets that have been washed with a solution of
(1) an amino acid which is glycine or alanine; PA1 (2) glycol, glycerol or methanol; PA1 (3) sodium chloride and sodium phosphate; and PA1 (4) a solid polyethylene glycol (molecular weight 4000 to 20,000).
The proposed mechanism of this reaction shows that the amino group of the amino acid reacts with the aldehyde group so that further reaction, which includes cross-linking that leads to hardening and shrinking of the platelets, cannot occur.
It is suprising that in the present invention superior results are obtained by utilizing iodoacetamide, which has the formula ICH.sub.2 CONH.sub.2, and ADA which has the formula H.sub.2 NCOCH.sub.2 N(CH.sub.2 COOH).sub.2, without using aldehyde-treated platelets.
Following the procedure of our invention, the long term stability is increased without changing the size distribution of the cells present. Thus the patient results which are reported to the diagnostic clinician can be assured of correct results. This leads to improved health care and improvements in the health care industry.